CONGENITAL: BASIC SCIENCE: REGENERATIVE THERAPIES

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Preclinical study of patient-specific cell-free nanofibertissue-engineered vascular grafts using 3-dimensionalprinting in a sheep model

Takuma Fukunishi, MD,a Cameron A. Best, BA,b Tadahisa Sugiura, MD, PhD,b Justin Opfermann, MS,c

Chin Siang Ong, MD,a Toshiharu Shinoka, MD, PhD,b Christopher K. Breuer, MD,b Axel Krieger, PhD,c

Jed Johnson, PhD,d and Narutoshi Hibino, MD, PhDa

ABSTRACT

Background: Tissue-engineered vascular grafts (TEVGs) offer potential to over-come limitations of current approaches for reconstruction in congenital heart dis-ease by providing biodegradable scaffolds on which autologous cells proliferateand provide physiologic functionality. However, current TEVGs do not addressthe diverse anatomic requirements of individual patients. This study exploresthe feasibility of creating patient-specific TEVGs by combining 3-dimensional(3D) printing and electrospinning technology.

Methods: An electrospinning mandrel was 3D-printed after computer-aideddesign based on preoperative imaging of the ovine thoracic inferior vena cava(IVC). TEVG scaffolds were then electrospun around the 3D-printed mandrel.Six patient-specific TEVGs were implanted as cell-free IVC interpositionconduits in a sheep model and explanted after 6 months for histologic, biochem-ical, and biomechanical evaluation.

Results: All sheep survived without complications, and all grafts were patentwithout aneurysm formation or ectopic calcification. Serial angiographyrevealed significant decreases in TEVG pressure gradients between 3 and6 months as the grafts remodeled. At explant, the nanofiber scaffold wasnearly completely resorbed and the TEVG showed similar mechanicalproperties to that of native IVC. Histological analysis demonstrated anorganized smooth muscle cell layer, extracellular matrix deposition, andendothelialization. No significant difference in elastin and collagen contentbetween the TEVG and native IVC was identified. There was a significantpositive correlation between wall thickness and CD68þ macrophage infiltrationinto the TEVG.

Conclusions: Creation of patient-specific nanofiber TEVGs by combiningelectrospinning and 3D printing is a feasible technology as future clinical option.Further preclinical studies involving more complex anatomical shapes arewarranted. (J Thorac Cardiovasc Surg 2017;153:924-32)

From the aDepartment of Cardiac Surgery, Johns Hopkins Hospital, Baltimore, Md;bTissue Engineering and Surgical Research, Nationwide Children’s Hospital;cThe Sheikh Zayed Institute for Pediatric Surgical Innovation, Children’s National

Medical Center, Washington, DC; and dNanofiber Solutions Inc, Columbus, Ohio.

This study was supported by grants from the National Institutes of Health Center for

Accelerated Innovations: Technology Development Program (grant No.

1UH54HL119810-01) and Thrasher Research Fund Early Career Awards (to

N.H.). Graft materials for this study were supplied by Nanofiber Solutions.

Read at the 42nd Annual Meeting of The Western Thoracic Surgical Association,

Waikoloa, Hawaii, June 22-25, 2016.

Received for publication J

publication Oct 13, 201

Address for reprints: Naru

Johns Hopkins Univers

(E-mail: nhibino1@jhm

0022-5223/$36.00

Copyright� 2016 Publish

for Thoracic Surgery

http://dx.doi.org/10.1016/

924 The Journal of Thoracic and Cardiovascular Surgery c April 2017

Study design of cell-free patient-specific nanofiber tissue

engineered vascular graft. A and B, Designing the graft

from a preoperative angiogram in the sheep model. C,

An electrospinning mandrel was modeled by computer

aided design and subsequently 3D-printed. D, Thenano-

fiber graft was electrospun onto the 3D printed mandrel.

E and F, The graft was implanted in sheep model.

j

Central Message

Our integrated approach of recent progress in 3D im-

aging, 3D printing, and tissue engineering technology

can create presurgically designed patient-specific

vascular grafts.

Perspective

Despite advances in surgical management for congen-

ital heart disease, a significant source of mortality

arises from surgical complexity. Patient-specific graft

design before surgery may yield benefits for quality of

life. This study demonstrates our integrated approach

of 3D imaging/printing, and tissue engineering tech-

nology can create presurgically designed patient-

specific vascular grafts.

See Editorial Commentary page 933.

See Editorial page 923.

une 28, 2016; revisions received Oct 12, 2016; accepted for

6; available ahead of print Dec 6, 2016.

toshi Hibino, MD, PhD, Department of Cardiac Surgery,

ity, 1800 Orleans St, Zayed 7107, Baltimore, MD 21287

i.edu).

ed by Elsevier Inc. on behalf of The American Association

.jtcvs.2016.10.066

Abbreviations and Acronyms3D ¼ three-dimensionalHPF ¼ high-power fieldIVC ¼ inferior vena cavaPGA ¼ polyglycolic acidPLCL ¼ poly(L-lactide-co-ε-caprolactone)SMC ¼ smooth muscle cellTCPC ¼ total cavopulmonary connectionTEVG ¼ tissue-engineered vascular graft

Scanning this QR code will takeyou to a video for the article.

Fukunishi et al Congenital: Basic Science: Regenerative Therapies

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Surgical treatment of many complex congenital cardiacanomalies involves implantation of synthetic conduits ofmaterials such as Gore-Tex and Dacron. A commonexample of such an application is the total cavopulmonaryconnection (TCPC) for single ventricle anomalies. Insome instances, the use of these synthetic grafts as conduitsis complicated by progressive obstruction, susceptibility toinfection, and risk of thromboembolic complications.1,2 Inall instances, a significant limitation to cardiovascularreconstruction is lack of growth potential of syntheticimplants. For TCPC, this may lead to suboptimalmanagement strategies, including either postponement ofcompletion of the Fontan circulation because of patientsize, or oversizing of conduits, which results insuboptimal flow characteristics, such as expiratory-phasebackflow and regions of flow stagnation.3

Tissue-engineered vascular grafts (TEVGs) offer thepotential to overcome these problems by providing abiodegradable scaffold in which autologous cells proliferateand mature into a physiologically functional blood vessel asscaffold polymers degrade.4-6 However, current TEVGs donot directly address the diverse anatomic and physiologicalrequirements of individual patients.

Imaging technologies such as computed tomographyand magnetic resonance imaging provide surgeons withdetailed, 3-dimensional (3D) views of complex cardiovas-cular anatomies before surgery. We have developed noveltechnology to create a patient-specific TEVG usingFood and Drug Administration–approved biodegradablenanofiber materials coated around a 3D printed mandrel(Figure 1).

In this study, we validated our novel technology byevaluating neotissue formation, biocompatibility, andmechanical properties of 3D-printed custom-made TEVGs

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after 6 months of implantation in a sheep model ofhigh-flow, low-pressure circulation.

MATERIALS AND METHODSPresurgery Imaging and 3D Model/MandrelCreation

Based on preoperative angiography images, the diameter and length of

the sheep inferior vena cava (IVC) were measured and matching

graft models were designed using computer-aided design software

(Solidworks, Waltham, Mass). The final mandrel design was converted

to stereolithography format and exported to Shapeways (New York, NY)

for 3D fabrication out of 420 stainless steel (Figure 2).

Scaffold FabricationTo create the co-electrospun polyglycolic acid (PGA) and

poly(L-lactide-co-ε-caprolactone) (PLCL) scaffolds, 10 wt% PGA and 5

wt% PLCL were dissolved in hexafluoroisopropanol. Each solution was

stirred via a magnetic stir bar for at least 3 hours at room temperature. In

separate syringes, the PGA solution was dispensed at a flow rate of

2.5 mL/hour, and the PLCL solution was dispensed at a flow rate of

5.0 mL/hour to create a graft with a 1:1 PGA:PLCL ratio. Both solutions

were simultaneously electrospun onto the custom 3D-printed mandrel

that was positioned 20 cm from the needle tip and rotated at 30 rpm. A

þ25-kV charge was applied to each syringe tip, and electrospun nanofibers

were deposited onto the grounded mandrel until the desired wall thickness

was achieved. The electrospun scaffold was then removed from the

mandrel, and thewall thickness was measured with a snap gauge by placing

the scaffold between 2 glass slides. The PGA/PLCL tubes were cut into

1.5-cm lengths, and the inner diameter was 12 mm. The scaffolds were

then packaged in Tyvek pouches and terminally sterilized with gamma

irradiation (Figure 2).

Mechanical TestingCompliance and burst pressure data were acquired using a universal

mechanical testing machine (MTS Systems, Eden Prairie, Minn), as

described previously.7 In brief, data were acquired using a load frame fitted

with a 50-pound load cell with a force resolution of 10�4 pounds and a

linear displacement resolution of 10�8 inches. Compliance testing was

performed using a displacement velocity of 1.5 mm/minute and an

acquisition rate of 4 data points/second using Laplace’s Law8,9 to

correlate linear force and displacement to compliance. Burst pressure

testing was performed using a displacement velocity of 50 mm/minute

and an acquisition rate of 4 data points/second using Laplace’s Law8,10

to correlate linear force and displacement to burst pressure.

Ring samples were placed around 2 parallel L-shaped steel rods. One

rod was attached to the base of the testing machine, and the other was

attached to the load cell. The samples were strained perpendicular to the

length of the sample. Compliance was calculated using systolic and

diastolic pressures of 120 mm Hg and 80 mm Hg, respectively. Burst

pressure was calculated as the maximum pressure immediately preceding

failure.

Graft ImplantationThe Animal Care and Use Committee at Q-Test Laboratories

(Columbus, Ohio) approved the care, use, and monitoring of animals for

these experiments. Six custom-made cell-free nanofiber TEVGs were

implanted as IVC interposition grafts in sheep (mean body weight,

23.9 � 5.0 kg). The animal size increased by roughly 2-fold over the

6-month study interval (mean body weight, 46.2 � 4.2 kg). All sheep

were anesthetized with 1.5% isoflurane during surgery. The IVC was

exposed, and heparin (100 IU/kg) was administered intravenously. The

TEVG was implanted as a supradiaphragmatic IVC interposition graft

rdiovascular Surgery c Volume 153, Number 4 925

FIGURE 1. Flow chart depicting the proposed process for manufacturing patient-specific tissue-engineered vascular grafts. A, Segmenting 3D image of the

vasculature. B, Creating a patient-specific graft design from the preoperative computed tomography image using the computer-aided design system.

C, Finalized mandrel. D, 3D-printed mandrel from stainless steel. E, Patient-specific nanofiber graft.

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using standard running 6-0 Prolene suture (Figure 2). Two surgical clips

were placed on the proximal and distal anastomoses as reference markers

for angiography and necropsy. Native supradiaphragmatic IVC served as a

control group, because the sheep supradiaphragmatic IVC is longer

than the human IVC, and it is located far away from right lung with no

surrounding tissues. Antibiotic treatment (cefazolin) was administered

intraoperatively and 7 days postoperatively. All sheep were maintained

on a daily oral dose of aspirin (325 mg/day) until the 6-month endpoint.

AngiographyAngiography was performed to assess any potential graft complications

at the 3- and 6-month time points. A 5 Fr catheter was inserted into the

jugular vein to the IVC, and intravenous contrast was manually -injected

FIGURE 2. Study design of cell-free patient-specific nanofiber tissue engineere

vena cava (IVC) was measured from angiography before surgery in the sheep

design and subsequently 3D-printed. D, The nanofiber scaffold was electrospun

tissue-engineered vascular graft (TEVG) was implanted as an IVC interposition

of the scaffold (G, 5003; H, 40003). I, Intraoperative picture of the implanted

926 The Journal of Thoracic and Cardiovascular Surg

into the supradiaphragmatic IVC and midgraft. In addition, IVC blood

pressure was measured at the proximal and distal anastomoses to evaluate

the pressure gradients across the graft.

Histology and ImmunohistochemistryExplanted TEVG samples were fixed in 10% formalin for 24 hours at

4 �C, and then embedded in paraffin. For standard histology, tissue sections

were stained with hematoxylin and eosin, Masson trichrome, picrosirius

red, Hart, and von Kossa stains. For immunohistochemistry, tissue sections

were deparaffinized, rehydrated, and blocked for endogenous peroxidase

activity and nonspecific staining. The primary antibodies used included

vonWillebrand factor (1:2000; Dako, Carpinteria, Calif), a-smoothmuscle

actin (1:500; Dako), myosin heavy chain (1:500; Abcam, Cambridge,

d vascular graft. A and B, The dimension and shape of the thoracic inferior

model. C, An electrospinning mandrel was modeled by computer-aided

onto the 3D-printed mandrel. E and F, A patient-specific cell-free nanofiber

conduit in the sheep model. G and H, Scanning electron microscope images

TEVG. 3D, 3-dimensional.

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FIGURE 3. Mechanical properties. The burst pressure (A) and compliance (B) of the native inferior vena cava (IVC) and patient-specific nanofiber

tissue-engineered vascular graft (TEVG) both before and at 6 months after implantation. Therewas no significant difference in burst pressure and compliance

between the native IVC and patient-specific nanofiber TEVG at 6 months. *P<.05.

Fukunishi et al Congenital: Basic Science: Regenerative Therapies

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Mass), and CD68 (1:200, Abcam). Antibody binding was detected using

biotinylated secondary antibodies (Vector Laboratories, Burlingame,

Calif), followed by incubation with streptavidinated horseradish

peroxidase (Vector Laboratories). Development was performed by

chromogenic reaction with 3,3-diaminobenzidine (Vector Laboratories).

Nuclei were counterstained with Gill’s hematoxylin (Vector Laboratories).

Histological and Quantitative AnalysisThe lumen diameter, wall thickness, remaining scaffold area, and

collagen content were measured from hematoxylin and eosin and

picrosirius red (polarized light) stainings using ImageJ software (National

Institutes of Health, Bethesda, Md). Picrosirius red staining revealed

collagen fibers.11 CD68þ macrophages were quantified by analyzing 4

high- power fields (HPFs; 403) from a representative section of each

sample (n ¼ 6) and averaged.

Biochemical AnalysisElastin content was determined using a Fastin colorimetric assay

(Biocolor, Carrickfergus, United Kingdom). For this assay, 100 mg dry

weight of each sample was measured and transferred to 1.5-mL

microcentrifuge tubes containing 750 mL of 0.25 M oxalic acid. The tubes

were then placed on a heat block for 60 minutes at 100�C to convert

insoluble elastin to water-soluble a-elastin. The elastin content in each

sample was determined by detection at 513 nm and extrapolation to a

standard curve after precipitation and dye binding following the

manufacturer’s protocol.

Collagen content was determined by a Sircol colorimetric assay

(Biocolor). For this, 100 mg dry weight of each sample was measured

and transferred to low-protein-binding 1.5-mL conical microcentrifuge

tubes containing 1.0 mL of pepsin (Sigma-Aldrich, St Louis, Mo), with a

concentration of 0.1 mg/mL of 0.5 M acetic acid to solubilize the collagen

bymeans of overnight incubation. The collagen content in each samplewas

determined by detection at 555 nm and extrapolation to a standard curve

after precipitation and dye binding following the manufacturer’s protocol.

StatisticsFor all experiments, data are represented graphically as scatterplots of

individual values with a bar identifying the median; manuscript references

are mean � standard deviation. Burst pressure, compliance, and outer

diameter change data were analyzed via one-way ANOVA with Tukey’s

multiple-comparisons test. Pressure gradient data were analyzed using

the paired 2-tailed t test. Analysis of elastin, collagen, collagen area %,

and wall thickness data was performed with the unpaired 2-tailed t test.

The significance of any correlation between wall thickness and CD68þ

macrophages/HPF was determined by calculating the Pearson correlation

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coefficient. A P value < .05 was considered statistically significant.

Statistical analysis was performed using Prism version 6 (GraphPad

Software, La Jolla, Calif).

RESULTSMechanical Properties of Patient-Specific Cell-FreeNanofiber TEVGsElectrospinning created a tubular scaffold with a uniform

wall thickness of 657 � 36 mm, significantly less than thenative IVC wall thickness of 1365 � 476 mm. Scanningelectron microscopy images of the scaffold are shown inFigure 2.There was no significant difference in the burst pressure

of the graft before implantation compared with native IVC.Similarly, no significant difference in burst pressure wasobserved between the native IVC and the TEVG at 6months(Figure 3, A; native IVC, 13,062� 6847mmHg vs 6-monthTEVG, 11,685 � 11,506 mm Hg vs preoperative TEVG,6167 � 5627 mm Hg; P ¼ .22). Preoperative graftcompliance was significantly greater compared with nativeIVC (Figure 3, B; preoperative TEVG, 4.0% � 1.5% vsnative IVC, 2.4% � 0.85%; P<.05); however, there wasno significant difference in compliance between nativeIVC and TEVG after 6 months (Figure 3, B; native IVC,2.4% � 0.85% vs 6-month TEVG, 2.3% � 0.46%;P>.05).

Biocompatibility in High-Flow Low-Pressure VenousCirculationGraft biocompatibility, including patency and tissue

remodeling, was excellent throughout the 6-month durationof the study. All sheep survived until the study endpointwith no graft-related complications, such as stenosis,dilation, or rupture (Figure 4, A). Native IVC and thepatient-specific TEVG were evaluated at 3 and 6 monthspostoperation for evidence of stenosis or dilation withcontrast-enhanced angiography. There was no significantdifference in the fold change of measured lumen diametersbetween 3 and 6 months when comparing the native IVC

rdiovascular Surgery c Volume 153, Number 4 927

FIGURE 4. Angiographic and quantitative biochemical analyses. A, Angiography of the patient-specific nanofiber tissue-engineered vascular grafts

(TEVGs) at 6 months. *Indicates right atrium; arrows indicate anastomosis site of graft. B, Angiographic diameter change over time indicating narrowing

of proximal, middle, and distal regions of patient-specific nanofiber TEVGs compared with native inferior vena cava (IVC) (6M/3M diameter). C, Therewas

a statistically significant difference in TEVG pressure gradient between the 3- and 6-month time points. **P ¼ .0045. D and E, The elastin and collagen

content of the TEVG was not significantly different from the native IVC.

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with the proximal, middle, or distal regions of the TEVGs.Both the IVC and patient-specific TEVG displayed nosignificant diameter changes between the 3- and 6-monthtime points (Figure 4, B). The pressure gradient across theTEVG at 6 months was significantly less than at 3 months,suggesting advantageous remodeling and scaffolddegradation (Figure 4, C; 3 months, 6.3 � 2.0 mm Hg vs6 months, 2.1 � 2.2 mm Hg; P ¼ .0045).

Elastin and collagen are critical for venous function andare well-studied markers of vascular graft remodeling.Biochemical quantification revealed the TEVG’selastin content (Figure 4, D; 6.7 � 3.1 mg/mg vs7.4 � 0.88 mg/mg; P ¼ .74) and collagen content(Figure 4, E; 1.8 � 0.4 mg/mg vs 1.8 � 0.35 mg/mg;P ¼ .93) were equivalent to that of the native ovine IVC.

Well-Organized Vascular Neotissue FormationThe tailor-made nanofiber TEVG demonstrated

well-organized vascular neotissue formation over 6 months.Hematoxylin and eosin staining demonstrated extensivecellular infiltration into the TEVG, evidence that scaffoldparameters, such as pore size and fiber diameter, permittedhost cell infiltration (Figure 5, E). All cell-free TEVGsremained patent without complications. Importantly,hematoxylin and eosin staining visualized under polarized

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light microscopy revealed that only 2.09% � 0.69% ofthe nanofiber scaffold material remained at 6 months,indicating that the vascular neotissue was the primarycontributor to the biochemical and mechanical propertiesassessed, further supporting the safety and efficacy of thisapproach. Furthermore, in vitro studies demonstrated thatthe TEVG loses all mechanical strength after 12 weeks(data not shown).

On the graft’s luminal surface, a cellular monolayerstained positively for von Willebrand factor, confirmingsuccessful endothelialization (Figure 5, F), like nativetissue (Figure 5, B). The luminal surfaces displayed noevidence of microthrombosis.

Smooth muscle cells (SMCs) are important for vascularfunction, and we identified mature contractile vascularSMCs using a-smooth muscle actin (Figure 5, G) andmyosin heavy chain markers at 6 months (Figure 5, H) inthe TEVG. A layer of myosin heavy- chain positive cellswas circumferentially organized and maintained adequatewall thickness at subintimal layers (Figure 5, H). Amultilayered population of a-smooth muscle actin–positivecells was present primarily in the neomedia, suggesting thatthe TEVG might not have been a completely matureneo-IVC at 6 months, and that active vascular remodelingwas still occurring at this time point (Figure 5, G).

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FIGURE 5. Representative hematoxylin and eosin photomicrographs of native inferior vena cava (IVC) (A) and patient-specific nanofiber

tissue-engineered vascular graft (TEVG) (E). Only 2.09% � 0.69% of the nanofiber scaffold material remained at 6 months. The TEVG endothelial cells

and smoothmuscle cells were mature and well-organized, and the smoothmuscle cell layer was thicker than the native IVC in area, distribution, and density.

Representative photomicrographs are shown for von Willebrand factor (B, native IVC; F, TEVG), a-smooth muscle actin (C, native IVC; G, TEVG), and

myosin heavy chain (D, native IVC; H, TEVG). Scale bars: 2 mm for A and E; 20 mm for B and F; 100 mm otherwise.

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Alternatively, a-smooth muscle actin could indicatesynthetic SMCs or myofibroblasts, and the a-smoothmuscle actin–positive/myosin heavy chain–negativestaining observed in the TEVG may identify the neo-adventitia or areas of continued growth and remodeling.

Extracellular matrix constituents in the TEVGmimicked the circumferential orientation observed inthe native IVC. The extracellular matrix density in theTEVG (Figure 6, E, picrosirius red; F, Masson’s tri-chrome) resembled that of native tissue (Figure 6, A,picrosirius red; B, Masson’s trichrome). Collagen deposi-tion, organization, and maturation were confirmed by as-sessing relative amounts and orientation of thin andthick fibers. The picrosirius red staining showed thatthe area fraction of collagen was similar in both theTEVG and native IVC (Figure 6, I; 9.75 � 6.27 vs

FIGURE 6. Collagen and elastin deposition in the tissue-engineered vascular g

(IVC) (A-D) without ectopic calcification. Representative photomicrographs for

von Kossa (D and H) stainings. Scale bars: 100 mm. I, Collagen content was si

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8.75% � 1.31%; P ¼ .74). Hart staining demonstratedthat the elastin composition of the TEVG (Figure 6, G)was comparable to that of the native IVC (Figure 6, C).There was no evidence of ectopic calcification in vonKossa staining at 6 months (Figure 6, D, native IVC;H, TEVG).The wall thickness of the TEVG was significantly larger

than that of native IVC (Figure 7, A; TEVG [n ¼ 6],2.27 � 0.91 mm vs native IVC [n ¼ 5], 0.89 � 0.15 mm;P ¼ .0091). We found a significant positive correlationbetween TEVG wall thickness and macrophageinfiltration into the scaffold (Figure 7, B, 2-tailedPearson correlation; P ¼ .0037; R2 ¼ 0.90), suggestingthat CD68þ macrophages induced the inflammatoryprocess of vascular remodeling in the graft, which

raft (TEVG) (E-H) at 6 months is similar to that of native inferior vena cava

picrosirius red (A and E), Masson’s trichrome (B and F), Hart’s (C, G), and

milar in both the TEVG and native IVC.

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FIGURE 7. Macrophage analysis and histomorphometry. A, Histomorphometric comparison of the wall thickness between native inferior vena cava (IVC)

and patient-specific nanofiber tissue-engineered vascular graft (TEVG) revealed a statistically significant difference. **P¼ .0091. B, In addition, therewas a

significant positive correlation between the wall thickness of patient-specific nanofiber TEVGs and CD68þ macrophages/high-power field (HPF) in the

patient-specific nanofiber TEVGs. P ¼ .0037. C, Macrophage infiltration into the TEVGs was assessed by manual quantification of CD68þ cells. Scale

bar: 20 mm.

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resulted in increased wall thickness of the TEVG(Figure 7, A).

DISCUSSIONThe results of this study validate the efficacy of

patient-specific, cell-free nanofiber TEVGs created bycomputer-aided design modeling, 3D printing, andelectrospinning. The patient-specific TEVG was compara-ble to native IVC in terms of mechanical properties,angiography, histology, and immunohistochemistry. Serialangiography revealed that the initial pressure gradientsbetween the cell-free, patient-specific TEVG and nativeIVC gradually resolved during the study’s 6-month timecourse as the polymer scaffold was resorbed. In addition,the mechanical profile of the TEVG resembled that of thenative IVC by the study endpoint.

Advances in 3D printing technologies are beingincreasingly integrated with the fields of regenerative andtranslational medicine to create novel solutions tochallenges in tissue engineering. One example of this isthe clinical use and development of a 3D-printedbiodegradable tracheal splint.12 The relevance of 3D-print-ing technologies in clinical applications of TEVGs has beenlimited, however, owing to a lack of suitable materials thatcan be 3D-printed. Recent studies have focused on fullysynthetic biodegradable polymers or synthetic polymersblended with natural proteins, such as collagen, elastin,gelatin, and chitosan.13-16 Although more currentbioprinting efforts have produced biological bloodvessels,17-19 the construct’s mechanical properties areinsufficient unless the tissue is further cultured formaturation. Previous studies also have examined TEVGfabrication using solvent-cast molding processes withsynthetic biodegradable polymers, but 3D-printing TEVGscaffolds would best streamline the process of creating apatient-specific conduit.20,21

We recently demonstrated the functionality of a3D-printed TEVG scaffold in a mouse model. The TEVG

930 The Journal of Thoracic and Cardiovascular Surg

was an unseeded, cell-free construct and had adequatemechanical properties capable of supporting vascular tissuegrowth both in vitro and in vivo.22 The 3D-printed TEVGswere implanted as IVC conduits in immunocompromisedmice and demonstrated biocompatibility and long-termefficacy at 1 year after the operation; however, thedegradation rate of this scaffold [poly(propylene fumarate)]was suboptimal. In this work, we used Food and DrugAdministration–approved materials with a known degrada-tion profile, a 3D-printed mandrel, and electrospinning toproduce custom TEVGs that could be easily translated toclinical application. Electrospinning is advantageous inthat it is a highly tunable process by which a wide varietyof polymer types and fiber sizes can be spun into variousshapes of mandrels, thereby allowing for the rational designof custom-made scaffolds for tissue engineering.23

The current ‘‘one-size-fits-all’’ paradigm inadequatelyaddresses the variable and complex anatomies present inthe congenital heart disease population. Specifically,patient-specific TEVGs are useful for patients withsingle-ventricle anomalies who undergo extracardiacTCPC. Clinical studies have demonstrated that thegeometry of the TCPC is a key factor in energy lossesfrom suboptimal hepatic flow distribution. In addition,advances in computational models have led to thedevelopment of more physiologically realistic patient-specific simulations.24 Computational modeling hasindicated that TCPC energy loss inversely correlates withdecreased diameter of the TCPC conduit and pulmonaryartery. These simulations also found correlations betweenhepatic flow distribution and caval offset, pulmonary flowdistribution, and the connection angle between the TCPCand SVC.25 Further studies have examined Y-Fontan graftsto avoid flow collision from the SVC and IVC and promoteequally distributed hepatic flow to the lungs.26 Althoughthese studies may be limited by their retrospective nature,the number of institutions involved, and hypotheticalcomputational simulations of rigid vessel walls, our

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VIDEO 1. Preclinical study of patient-specific cell-free nanofiber tissue-

engineered vascular grafts (TEVGs) using 3D printing in a sheep model.

Despite advances in surgical management of congenital heart disease, sig-

nificant morbidity and mortality arise due to surgical complexity. Patient-

specific graft design before surgery has the potential to overcome these

challenges and improve patients’ quality of life. This study demonstrates

our integrated approach of 3D imaging, 3D printing, and tissue engineering

technology to design and create patient-specific vascular grafts before sur-

gery in a sheep model. There were no significant differences in burst pres-

sure and compliance between grafts and native inferior vena cava (IVC).

The grafts showed native-like neotissue formation at 6 months after surgery

with their scaffolds almost fully degraded, with only 2% of the initial scaf-

folds remaining on average. Extracellular matrix, elastin, and collagen pro-

duction and endothelialization were similar in grafts and native IVC, with

no vascular calcification. The presence of multilayer smooth muscle cells

and a decreasing pressure gradient at 6 months compared with 3 months

suggested graft remodeling. There was a significant positive relationship

between graft wall thickness and CD68þ macrophage infiltration into the

scaffold. We have demonstrated the feasibility of creating patient-

specific nanofiber TEVGs using combined technology of electrospinning

and 3D printing. Video available at: http://www.jtcvsonline.org/article/

S0022-5223(16)31466-0/addons.

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patient-specific concept is able to address or altogethereliminate several of the concerns raised regardingtraditional approaches to the Fontan circulation. The nano-fiber construct evaluated in this report promotes autologousvessel growth with the potential of overcoming concernsassociated with more rigid materials, such as flow and diam-eter mismatch. Patient-specific grafts can also avoid theneed to offset SVC positioning in patients with limited im-plantation space owing to anatomic restrictions. Therefore,the patient-specific TEVG, by taking advantage of recentadvances in imaging and computational modeling, providesimmense potential to provide improved clinical and surgicaloutcomes. We suggest that our data support the continuedpursuit of creating a complex, bifurcated, personalizedTEVG for use as an optimized TCPC connection.

A patient-specific nanofiber scaffold is perhaps the nextnatural evolution of the TEVG. TEVGs in the form ofbiodegradable scaffold (woven PGA reinforced withPLCL) have been safely applied in low-pressureenvironments for patients with congenital heart diseaseundergoing extracardiac TCPC.4-6 Using routineradiographic modalities for TEVG observation, long-termfollow-up demonstrated no instances of graft-relatedmortality and identified no cases of aneurysm formation,graft rupture, or ectopic calcification.27 Although the choiceof PLCL and PGA as synthetic biodegradable materialshas led to effective graft mechanical properties andremodeling in the previous human trial, we are currentlyevaluating second-generation electrospun PGA/PLCL andPCL/chitosan28 scaffold designs with optimized nanofiberparameters that do not require cell seeding.

The patient-specific nanofiber TEVG displayedsatisfactory vascular remodeling at 6 months postoperation.To the best of our knowledge, we are the first to demonstratevascular SMC maturation in a biodegradable electrospunnanofiber scaffold in a sheep model. Compared with nativeIVC, the TEVG at 6 months had a significantly thicker layerof SMCs. Angiography showed significantly lower pressuregradients of the TEVG at 6 months than at 3 months. Inaddition, there was no significant difference at 6 monthsbetween the TEVG and native IVC in terms of burstpressure and compliance. A well-organized vascular SMClayer is required to prevent vascular calcification, butexcessive SMC proliferation may eventually lead tostenosis or complete occlusion owing to hyperplasia. The6-month time course of this study makes it difficult toascertain whether the vascular SMC layer that developedin the TEVG would eventually resemble native IVC orprogress to become occlusive stenosis at some later timepoint.

With regard to an adverse immune response at 6 months,we found a significant positive correlation between TEVGwall thickness and CD68þ macrophage infiltration intothe scaffold. We previously reported that a scaffold seeded

The Journal of Thoracic and Ca

with bone marrow mononuclear cells transforms into aliving vascular conduit with the ability to grow, repair,and remodel via an inflammation-mediated process.29

Additionally, our previous studies suggested that excessivemacrophage infiltration contributes to occlusive vascularneotissue hyperplasia.30,31 Compared with native IVC,TEVG wall thickness was thicker, which may indicate theprogression of chronic intimal hyperplasia.Although the results of this study are promising and

clearly encourage further investigation, this study waslimited by its short in vivo time course and small samplesize. Although creating a single ventricular model is morechallenging in vivo, as is done clinically with theextracardiac TCPC procedure, future studies should beperformed with complex anatomies. In addition, eventhough our scaffold demonstrated almost completedegradation in our previous in vivo studies, the degradationprofile of the nanofiber TEVG should be further evaluated

rdiovascular Surgery c Volume 153, Number 4 931

Congenital: Basic Science: Regenerative Therapies Fukunishi et al

CONG

during long-term follow up before being applied to humanclinical trials. Despite our study limitations, patient-specificnanofiber TEVGs are an attractive alternative to prostheticvascular grafts, such as Gore-Tex and Dacron, andour patient-specific nanofiber TEVG warrants furtherinvestigation owing to its immense clinical potential(Video 1).

Conflict of Interest StatementAuthors have nothing to disclose with regard to commercialsupport.

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Key Words: 3D printing, cell-free tissue engineering,congenital heart disease, electrospun nanofibers, Fontancirculation, patient-specific, preclinical study, sheep model,tissue-engineered vascular graft

ery c April 2017